High resolution melting analysis as a prescreening tool

ABSTRACT

Compositions and methods for determining an increased likelihood of a response to a targeted treatment of a cancer disease including isolating genomic DNA from a patient sample, amplifying a fragment of DNA by means of PCR with a specific pair of amplification primers, determining if the amplified fragment comprises a wildtype sequence or a mutation by means of a High Resolution Melting Analysis (HRM), and correlating the presence or absence of a mutation with an increased likelihood of success of said targeted treatment. Respective primer pairs, compositions and kits are also claimed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of the pending application Ser. No.13/930,638 filed on Jun. 28, 2013 which is a continuation ofInternational Application No. PCT/EP2012/050213, filed Jan. 9, 2012,which claims the benefit of European Patent Application No. 11150641.6,filed Jan. 11, 2011, the disclosures of which are hereby incorporated byreference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 27, 2013, isnamed SEQUENCE_LISTING_(—)27206US.txt, and is six thousand nine hundredand ninety-five bytes in size.

BACKGROUND OF THE DISCLOSURE

Therapeutic agents targeting molecular changes are involved in thetreatment of several human cancers. The epidermal growth factor receptor(EGFR), a transmembrane tyrosine kinase receptor, is one of the targetsin colorectal cancer therapy because of its deregulation in 60% to 80%of the cases. Activated signaling pathways include theRAS/RAF/mitogen-activated protein kinase (MAPK) pathway and thephosphatidylinositol3-kinase (PI3K)/PTEN/AKT pathway involved in cellproliferation, angiogenesis, apoptosis and metastatic processes(Baselga, J., Eur. J. Cancer 37 Suppl 4 (2001) S16-S22).

Monoclonal antibodies targeting EGFR, like cetuximab (Erbitux®) andpanitumumab (Vectibix®) have entered clinical practice and have shownbenefit in approximately 10% to 20% of patients with colorectal cancersdue to the inhibition of downstream pathways (Amado, R. G., et al., J.Clin. Oncol. 26 (2008) 1626-1634; Cunningham, D., et al., N. Engl. J.Med. 351 (2004) 337-345; Saltz, L B., et al., J. Olin Oncol. 22 (2004)1201-1208; Van Cutsem, E., et al., J. Clin. Oncol. 25 (2007) 1658-1664).Previous work has shown that mutations in KRAS negatively correlate withthe response to anti-EGFR antibodies and therefore are an independentpredictive marker of resistance against this therapy (Lievre, A., etal., Cancer Res. 66 (2006) 3992-3995). Based on these results, theEuropean Medicines Agency (EMEA), has approved the use of panitumumaband cetuximab only for patients with metastatic colorectal cancerwithout activating mutations in KRAS and mutation analyses should bepart of the pretreatment. KRAS mutations account for 30% to 40% of thecases resistant to anti-EGFR therapies (Di Fiore, F., et al., Br. J.Cancer 96 (2007) 1166-1169; Lievre, A., et al., Cancer Res. 66 (2006)3992-3995). Some studies suggested that additional mutations concerningthe RAS/RAF/MAPK and PI3K/PTEN/AKT pathway are involved. Patients withwildtype KRAS and mutations in BRAF do not respond to anti-EGFR therapy,whereas wildtype BRAF status seemed to increase the therapy efficiency(Di Nicolantonio, F., et al., J. Olin. Oncol. 26 (2008) 5705-5712).Recently it was shown that BRAF mutations in colorectal cancer arerather of prognostic than predictive value (Tol, J., et al., Eur. J.Cancer 46 (2010) 1997-2009).

The BRAF V600E mutation is one of the most common mutations in humancancer with a high incidence in malignant melanoma (Curtin, J. A., etal., N. Engl. J. Med. 353 (2005) 2135-2147). High objective responserates in melanoma patients carrying this mutation were observed in thephase I clinical trial of the RAF inhibitor PLX4032 and phase II andphase Ill studies are limited to patients with BRAF V600E mutation(Flaherty, K. T., et al., N. Engl. J. Med. 363 (2010) 809-819). Recentlyit has been shown that patient bearing the BRAF V600K mutation respondremarkably to PLX4032, suggesting that mutation assays for codon 600should at least include the exchanges V600E and V600K (Bollag, G., etal., Nature 467 (2010) 596-599; Rubinstein, J. C., et al., J. Transl.Med. 8 (2010) 67).

PIK3CA mutations have been described in up to 40% of invasive breastcarcinoma, and AKT1 mutations have been found in up to 8% of breastcarcinoma (Dunlap, J., et al., Breast Cancer Res. Treat 120 (2010)409-418). These mutations occur early in breast cancer development andmay have implications on the selection of therapeutics targeting the PI3kinase pathway. Moreover it has been shown in lung cancer that combinedinhibition of activated PI3K and MAPK signaling might be clinicallybeneficial (Sos, M. L., et al., Proc. Natl. Acad. Sci. USA 106 (2009)18351-18356).

BRIEF SUMMARY OF THE DISCLOSURE

The present disclosure relates to the field of predicting susceptibilityfor certain tumor therapies by means of appropriate mutational analysisof the patient's genome. Embodiments of the instant application discloseHigh Resolution Melting (HRM) assays as a prescreening diagnostic methodto diagnose mutations in the hot spot regions of the most common genes(KRAS, BRAF, PIK3CA, AKT1) concerning the RAS/RAF/MAPK and PI3K/PTEN/AKTpathway.

According to some embodiments, the present disclosure provides pairs ofamplification primers, which are useful for HRM analysis of genes whichare important for predicting responsiveness to cancer therapeuticagents. In particular, the present disclosure provides the followingpairs of amplification primers for amplification and analysis of KRAS,exons 2 and 3, BRAF, exon 15, PIK3CA, exons 7, 9 and 20, and AKT1, exon2. More precisely, the present disclosure provides the following primerpairs:

Seq. ID. No: 1 and Seq. ID. NO: 2 AKT 1, exon 2 Seq. ID. No: 3 and Seq.ID. NO: 4 AKT1, exon 2 Seq. ID. No: 5 and Seq. ID. NO: 6 BRAF, exon 15Seq. ID. No: 7 and Seq. ID. NO: 8 BRAF, exon 15 Seq. ID. No: 9 and Seq.ID. NO: 10 KRAS, exon 2 Seq. ID. No: 11 and Seq. ID. NO: 12 KRAS, exon 2Seq. ID. No: 13 and Seq. ID. NO: 14 KRAS, exon 3 Seq. ID. No: 15 andSeq. ID. NO: 16 KRAS, exon 3 Seq. ID. No: 17 and Seq. ID. NO: 18 PIK3CA,exon 7 Seq. ID. No: 19 and Seq. ID. NO: 20 PIK3CA, exon7 Seq. ID. No: 21and Seq. ID. NO: 22 PIK3CA, exon 9 Seq. ID. No: 23 and Seq. ID. NO: 24PIK3CA, exon 9 Seq. ID. No: 25 and Seq. ID. NO: 26 PIK3CA, exon 20 Seq.ID. No: 27 and Seq. ID. NO: 28 PIK3CA, exon20

Embodiments of the present disclosure also provide a composition orreaction mixture comprising at least one pair of amplification primersas disclosed above. The composition may be used for PCR amplification ofnucleic acids during a nucleic acid amplification reaction, and also PCRamplification and monitoring in real time. According to some embodimentsof the present disclosure, a mixture comprises at least: a pair ofamplification primers as disclosed above; a thermostable DNA Polymerase;a mix of deoxynucleoside triphosphates which is usually dA, dG, dC anddT, or dA, dG, dC and dU; and a buffer.

In some further embodiments, when suitable for amplification anddetection in real time, of one or more specific nucleic acid targetsequence(s) such a composition additionally comprises a nucleic aciddetecting entity such as a fluorescent hybridization probe, or afluorescent, double stranded DNA binding dye. In some embodiments, sucha DNA double stranded Dye is a dye which can be used to perform HRMcurve analysis. Illustrative embodiments of such dye include theLightCycler® 480 Resolight dye (Roche Applied Science Cat. No: 04 909640 001) or similar compounds as disclosed in WO 2008/052742.

According to some illustrative embodiments of the instant disclosure,the pair of amplification primers is designed to amplify a specificsequence of interest according to standard methods known in the art ofmolecular biology. In some embodiments, when brought into contact with asample that shall be analyzed, such a PCR reaction mixture additionallycomprises an at least partially purified DNA or other nucleic acid whichputatively comprises a specific sequence of interest. Also, in some suchembodiments, the concentrations of all reagents included are generallyas known to persons skilled in the art and can be optimized for specificadaptations according to standard protocols. In some such embodiments,the concentration of the fluorescent, double stranded DNA binding dye isbetween approximately 0.1 to 10.0 μg/ml.

In some embodiments of the instant disclosure, a kit is provided. Someillustrative embodiments of kits disclosed herein include at least onepair of amplification primers as disclosed above and herein. Someembodiments of kits disclosed herein may further comprise one, several,or all of the following additional ingredients: a thermostable DNAPolymerase; a mix of deoxynucleoside triphosphates which is usually dA,dG, dC and dT, or dA, dG, dC and dU, and a buffer, and a fluorescent,double stranded DNA binding dye, which may be suited to be used for HRM.

In yet additional embodiments of the instant disclosure,oligonucleotides are provided which may be used as hybridization probesfor new nucleotide sequence variations with KRAS, for example whichhaven't been disclosed in the art. In some such embodiments, the presentdisclosure also provides an oligonucleotide comprising a sequenceselected from a group consisting of Seq. ID. NO: 31 or its complement,Seq. ID. NO: 32 or its complement, Seq. ID. NO: 33 or its complement,and Seq. ID. NO: 34 or its complement.

In even further embodiments, the present disclosure is directed to amethod for determining the increased likelihood of a response to atargeted treatment of a cancer disease, comprising the steps of:

-   -   a) isolating genomic DNA from a patient sample;    -   b) amplifying at least one fragment of said DNA by means of PCR        with a specific pair of amplification primers;    -   c) determining, whether said amplified fragment has a wildtype        sequence or comprises a mutation by means of a High Resolution        Melting Analysis (HRM); and    -   d) correlating the presence or absence of a mutation with an        increased likelihood of success of said targeted treatment.

According to some embodiments, the mutation is identified by means of ahybridization analysis or by means of sequencing. For example, thepatient sample may be Formalin Fixed Paraffin Embedded (FFPE) tissue. Insome such cases, HRM Analysis may be performed without any spiking ofDNA.

In some even further embodiments, the (at least one) fragment isselected from a group comprising KRAS, exon 2, KRAS, Exon 3, BRAF exon15, PIK3CA exon 7, PIK3CA exon 9, PIK3CA exon 20, and AKT1 exon 2. Insome such embodiments, at least one pair of amplification primersselected from the group consisting of: Seq. ID. No: 1 and Seq. ID. NO:2; Seq. ID. No: 3 and Seq. ID. NO: 4; Seq. ID. No: 5 and Seq. ID. NO: 6;Seq. ID. No: 7 and Seq. ID. NO: 8; Seq. ID. No: 9 and Seq. ID. NO: 10;Seq. ID. No: 11 and Seq. ID. NO: 12; Seq. ID. No: 13 and Seq. ID. NO:14; Seq. ID. No: 15 and Seq. ID. NO: 16; Seq. ID. No: 17 and Seq. ID.NO: 18; Seq. ID. No: 19 and Seq. ID. NO: 20; Seq. ID. No: 21 and Seq.ID. NO: 22; Seq. ID. No: 23 and Seq. ID. NO: 24; Seq. ID. No: 25 andSeq. ID. NO: 26; and Seq. ID. No: 27 and Seq. ID. NO: 28, is used.

BRIEF DESCRIPTION OF THE FIGURES

The features of this disclosure, and the manner of attaining them, willbecome more apparent and the disclosure itself will be better understoodby reference to the following description of embodiments of thedisclosure taken in conjunction with the accompanying drawing.

FIG. 1A-1F show a comparison of KRAS exon 2 (A-C) and PIK3CA exon 9(D-F) HRM assays using DNA isolated from cell lines (A and D) or fromFFPE tissues (B and E). All samples are displayed in triplicates. (A),(B):KRAS wildtype: HT-29, KRAS mutations: HCT116 (G13D, heterozygous),LoVo (G12V, homozygous), A549 (G12S, homozygous), RPMI 8226 (G12A,heterozygous), LS174T (G12D, heterozygous), Mia PaCa-2 (G12C,homozygous); and (D), (E): PIK3CA wildtype: HCT116, PIK3CA mutations:MCF-7 (E545K, heterozygous), NCI N417 (Q546K, heterozygous) and BT-20(P539K, heterozygous); (C), (F): Determination of the sensitivities ofKRAS exon 2 (C) and PIK3CA exon 9 (F) HRM mutations assays. Percentagesindicate the proportion of mutant DNA relative to wildtype DNA. 6.3% (C)and 3.1% (F) of mutated DNA isolated from HCT116 (G13D, heterozygous, C)or NCI N417 (Q546K, heterozygous, F) were detectable.

Corresponding reference characters indicate corresponding partsthroughout the several views. Although the drawings representembodiments of the present disclosure, the drawings are not necessarilyto scale and certain features may be exaggerated in order to betterillustrate and explain the present disclosure. The exemplifications setout herein illustrate an exemplary embodiment of the disclosure, in oneform, and such exemplifications are not to be construed as limiting thescope of the disclosure in any manner.

DETAILED DESCRIPTION OF THE DISCLOSURE

The embodiments disclosed herein are not intended to be exhaustive orlimit the disclosure to the precise form disclosed in the followingdetailed description. Rather, the embodiments are chosen and describedso that others skilled in the art may utilize their teachings.

Target specific therapies require specific and sensitive mutationscreening methods, which allow fast and high throughput analyses.Various methods have been described for the detection of mutations, suchas Sanger Sequencing (Yunxia, Z., et al., BMC Med. Genet. 11 (2010) 34),real-time PCR (Amicarelli, G., et al., Nucleic. Acids Res. 35 (2007)e131) and pyrosequencing (Ogino, S., et al., J. Mol. Diagn. 7 (2005)413-421).

Recently, High Resolution Melting (HRM) was introduced ed as a newmolecular technique for high throughput mutation scanning (Zhou, L, etal., Clin. Chem. 51 (2005) 1770-1777). For example, HRM instrumentation(Roche Applied science Cat. No: 05 015 278 001), software ((RocheApplied Science Cat. No: 05 103 908 001) and dyes (Roche Applied ScienceCat. No: 04 909 640 001) are commercially available. Mutationdetermination using HRM is based on the dissociation of DNA, whenexposed to an increasing temperature in the presence of fluorescent dyesinteracting with double-stranded DNA (see, for example U.S. Pat. No.7,387,887, U.S. Pat. No. 7,582,429). There are numerous appropriate dyesdisclosed in the art. The presence of a mutation leads to the formationof DNA heteroduplexes followed by a change in melting behaviour.

High Resolution Melting (HRM) is a mutation scanning” technique thatdetects the presence of sequence variations in target-gene derived PCRamplicons. HRM enables genomic researchers to analyze genetic variationsin PCR amplicons prior to or as alternative to sequencing by means of aclosed-tube post-FOR (Polymerase Chain Reaction) analysis method. HighResolution Melting provides high specificity, sensitivity andconvenience at significantly higher speed and lower cost than otherestablished (e.g., gel-based) methods. For example, in a diploid genome,equivalent regions from maternal and paternal chromosomes are bothamplified by the Polymerase Chain Reaction. The PCR products can then beanalyzed for completely matched hybrids (called homoduplexes) andmismatched hybrids (heteroduplexes). Thus, the entire mutation screeningprocess is homogeneous. High Resolution Melting, is a refinement ofearlier, well-established DNA dissociation (or “melting”) techniques(e.g., to determine the Tm of a DNA hybrid). Like all melting analyses,the technique subjects DNA samples to increasing temperatures andrecords the details of their dissociation from double-stranded (dsDNA)to single-stranded form (ssDNA).

Before a High Resolution Melting analysis can be performed, the targetsequence must be available in high copy number. The easiest way toaccomplish this is to perform a DNA amplification reaction (PCR) in thepresence of a High Resolution Melting Dye before the High ResolutionMelt. Prominent examples of such dyes are disclosed in WO 2008/052742.After PCR, the successive melting experiment can be performed on thesame Real Time Instrument, and analyzed with a respective Gene ScanningSoftware to identify sequence variants. As such, the entire experimentcan be done without opening the reaction vessels and without additionalhandling steps after the PCR setup. Both procedures are performed in thepresence of a fluorescent dye that binds only dsDNA. The dye does notinteract with ssDNA, but fluoresces strongly in the presence of dsDNA.This change in fluorescence can be used both to measure the increase inDNA concentration during PCR and then to directly measurethermally-induced DNA dissociation during High Resolution Melting.

For detection of sequence variations, differences in the melting curvesof the amplicons are analyzed. Heterozygote DNA forms heteroduplicesthat begin to separate into single strands at a lower temperature andwith a different curve shape than homozygote DNA. Depending on theindividual sequence, most of the different homozygotes givedistinguishable melting curves, too.

In a melting experiment, fluorescence is initially high because thesample starts as dsDNA, but fluorescence diminishes as the temperatureis raised and DNA dissociates into single strands. The observed“melting” behavior is characteristic of a particular DNA sample.Mutations in PCR products are detectable because they change the shapeof the melting curve. When the mutant sample is compared to a reference“wild type” sample, these changes are visible.

Usually, an analysis of HRM data is supported by a respective software,which, in some embodiments, advantageously provides for the followinganalysis steps:

Normalization:

The raw melting curve data can be normalized by setting the pre-melt(initial fluorescence) and post-melt (final fluorescence) signals of allsamples to uniform values. Pre-melt signals are uniformly set to arelative value of 100%, while post-melt signals are set to a relativevalue of 0%. Normalizing the initial and final fluorescence in allsamples aids interpretation and analysis of the data. In some cases,samples with homozygous SNPs may be distinguished from the wild type bythe displacement of their melting curves, which is easier to see inthenormalized data.

Temperature Shifting:

A shift on the temperature axis of the normalized melting curves at thepoint where the entire double-stranded DNA is completely denatured.

Difference Plot:

Further information on the differences in melting curve shapes,different can be obtained by means of subtracting the curves from areference curve (also called “base curve”), thus generating a DifferencePlot, which helps cluster samples automatically into groups that havesimilar melting curves.

Summarizing, High Resolution Melting (HRM) can be defined as a methodcomprising the steps of: monitoring temperature dependent fluorescenceof the dsDNA binding dye in order to generate a melting curve from apreviously amplified target nucleic acid; repeating the providing,amplifying, monitoring and generating steps with at least one additionalpreviously amplified target nucleic acid, and comparing the generatedmelting curves with each other.

Such a comparison may be achieved by means of creating a DifferencePlot. For example, amplification may be performed by means of PCR,during which said dsDNA binding dye is already present within the samplesuch that the whole assays can be performed in a homogenous manner.

As the availability for targeted therapies for several tumor typesincreases the need for rapid and sensitive mutation screening isgrowing. The aim of the inventors was to establish High ResolutionMelting (HRM) assays for routinely used predictive analyses of KRAS,AKT1, PIK3CA and BRAF mutations. In KRAS mutations constitutivelyactivate the RAS/RAF/mitogen-activated protein kinase (MAPK) pathway andtherefore play an important role in anti-EGFR therapy for patients withcolorectal cancers. Mutationally activated PIK3CA and AKT1 are promisingtherapeutic targets in breast cancer. In 60-70% of malignant melanoma amutation in BRAF can be found. Thus blocking the oncogenic signallinginduced by this mutation is employed now as treatment approach.

HRM assays were developed using genomic DNA containing the desiredmutation, enabling the detection of 3.1% to 12.5% mutated DNA mixed inwildtype background. For KRAS (exon 2) and PIK3CA (exon 9), assays wereproofed by hybridization probes and/or Sanger Sequencing to excludepseudogene amplification. The HRM assays were validated using genomicDNA isolated from formalin-fixed paraffin-embedded (FFPE) tissues fromdifferent tumor entities. Sanger Sequencing was used to confirm andcharacterize HRM results. The concordance between Sanger Sequencing andHRM was 99% for KRAS exon 2 and PIK3CA exon 20 and 100% for theremaining assays. In conclusion HRM provides a powerful tool to detectgenomic mutations efficiently.

High sensitivity, high specificity, transferability to other tumorentities and robustness in analysing low-quality genomic DNA isolatedfrom FFPE tissues, were highly important prerequisites for thedevelopment of new HRM mutation assays. As such, HRM assays wereestablished and optimized using DNA samples bearing the most frequentmutations in the genes of interest. For each at least three differentprimer sets were tested in order to find optimal conditions. Thesensitivity of each assay was assessed by testing dilutions of mutatedDNA strands in a wildtype background. Special attention was paid to theexclusion of pseudogene amplification. Finally all HRM assays werevalidated using a collection of FFPE tissues from various tumorentities. Mutation analyses from all FFPE tissues were additionallyconfirmed by Sanger Sequencing.

The development of targeted therapies and therefore the identificationof predictive markers is gaining importance in the field of moleculardiagnostics. Determination of genetic markers in several tumor entitiesallows individualized treatment of patients, as for example the KRASmutation analysis which helps to predict the response to anti-EGFRtherapy in patients with colorectal cancer (Amado, R. G., et al., J.Olin. Oncol. 26 (2008) 1626-1634; Lievre, A., et al., Cancer Res. 66(2006) 3992-3995).

The present disclosure provides high sensitive testing methods thatallow rapid identification of hot-spot mutations in the main genes(KRAS, BRAF, PIK3CA, AKT1) involved in the RAS/RAF/MAPK andPI3K/PTEN/AKT pathway.

The present disclosure discloses the use of HRM analysis as a reliableand sensitive prescreening technology to detect genomic changes in DNAisolated from FFPE tissues. Several methods used in the art to determinegenomic variations are described, for example Sanger Sequencing, thegold standard for mutational analysis, pyrosequencing or real-time-basedPCR analysis. The major disadvantage for real-time-based PCR analyses isthe need for expensive fluorescence-labeled probes. Pyrosequencingprovides a sensitive method, but may not be economical due to expensiveequipment (Pichler, M., et al., J. Mol. Diagn. 11 (2009) 140-147).Compared to that HRM is a cost effective method, which is at the sametime fast in contrast to Sanger Sequencing (Franklin, W A, et al., J.Mol. Diagn. 12 (2010) 43-50; Monzon, F. A., et al., Arch. Pathol. Lab.Med. 133 (2009) 1600-1606). HRM is highly applicable for large scalegenotyping because of its sensitivity and simplicity as well as the lowDNA amount required. It can be used as a prescreening method andfollowed by direct Sanger Sequencing of the HRM product when additionaldetermination of the exact mutation is needed. This saves manpower andresources and provides rapid results concerning all patients withoutmutations.

HRM analysis allows the detection of a wide spectrum of mutationswhereas the commercially available TheraScreen K-RAS Kit is only able todetect seven mutations in KRAS exon 2 (G12D, G12V, G12C, G125, G12A,G12R, G13D) targeted by the designed primers within the kit. In the caseof untargeted mutation the kit provides a false negative result (Angulo,B., et al., J. Mol. Diagn. 12 (2010) 292-299). Additionally to the sevenmain KRAS mutations, three mutations (G13C, G13_V14insG, V14A) could bedetected using the HRM assay which are not included in the TheraScreenK-RAS Kit. An insertion mutation of KRAS codon 12 (G12 G13insG) isalready published (Servomaa, K., et al., Mol. Pathol. 53 (2000) 24-30).The inventors have identified an insertion mutation of KRAS codon 13(G13_V14insG) and a point mutation (V14A) which are both to the best oftheir knowledge not described yet. However KRAS V141 mutations aredescribed in colorectal cancer (Ferraz, J. M., et al., Int. J. Cancer110 (2004) 183-187), myeloid leukemia (Tyner, J. W., et al., Blood 113(2009) 1749-1755) and in three patients with Noonan syndrome (Schubbert,S., et al., Nat. Genet. 38 (2006) 331-336). Functional assays exhibitedoncogenic properties of V141 mutation in comparison to wildtype KRAS(Tyner, J. W., et al., Blood 113 (2009) 1749-1755). Concerning KRAS exon3 HRM assay two new mutations (G60D, Q61L: c.182_(—)183AA>TG), which arenot described until now could be found. Altogether this emphasizes thenecessity to use a method, which allows the detection of all genomicvariations.

The sensitivities of developed HRM assays to detect genomic mutated DNAin wildtype background range from 3.1% to 12.5% and are comparable tosensitivity data described recently (Krypuy, M., et al., BMC Cancer 6(2006) 295) and similar to alternative methods like pyrosequencing (5%to 10% (Monzon, F. A., et al., Arch. Pathol. Lab. Med. 133 (2009)1600-1606)) or TheraScreen K-RAS Mutation Kit (5% (Angulo, B., et al.,J. Mol. Diagn. 12 (2010) 292-299)). Sanger Sequencing needs a largeramplicon size (about 250 bp) compared to HRM, resulting in a higherfailure rate due to the low quality of DNA isolated from FFPE tissue.Furthermore Sanger Sequencing shows a lower sensitivity of about 20-30%mutated DNA mixed with wildtype DNA (Monzon, F. A., et al., Arch.Pathol. Lab. Med. 133 (2009) 1600-1606; Pichler, M., et al., J. Mol.Diagn. 11 (2009) 140-147). Mutations found by HRM during the studyunderlying the present disclosure in the PIK3CA gene from three FFPEsamples (3/16) could only be confirmed by Sanger Sequencing using theHRM PCR products directly and not the standard approach. This increasein sensitivity may be due to the shorter amplicon size. Using the HRMPCR product for Sanger Sequencing is not always possible. One example isKRAS exon 3 where the forward primer is located too near to the regionof interest. The concordance between results from Sanger Sequencing andHRM analyses was 100% except for KRAS exon 2 and PIK3CA exon 20, whichhave 99% correlation. This could be due to the lower sensitivity of theSanger Sequencing and was comparable to recent publications (95% (Ma, E.S., et al., J. Clin. Pathol. 62 (2009) 886-891), 100% (Pichler, M., etal., J. Mol. Diagn. 11 (2009) 140-147)). HRM represents a suitablemethod for screening of low frequency mutations such as AKT1 exon 2 orPIK3CA exon 7 mutations in clinical samples.

Referring to recent publications, the amplicon size (the shorter thebetter), exclusion of primer dimers, salt concentration, specificmelting products with only one single melting domain and standardizedgenomic DNA isolation protocols are important for implementation ofhighly sensitive HRM assays (Pichler, M., et al., J. Mol. Diagn. 11(2009) 140-147; Reed, G. H., et al., Pharmacogenomics 8 (2007) 597-608;van Eijk, R., et al., J. Mol. Diagn. 12 (2010) 27-34). Performingtriplicates is necessary to minimize temperature differences on themicrotiter plate (Herrmann, M. G., et al., Clin. Chem. 52 (2006)494-503). By using highly sensitive hybridization probes and/or SangerSequencing to exclude KRAS or PIK3CA pseudogene amplification highspecificity of the established HRM assays could be confirmed. Shortamplicon sizes (100 bp to 183 bp) allow analyses of low quality DNAisolated from FFPE tissues and feasibility in routine diagnosticlaboratory is guaranteed.

HRM analysis detects changes in DNA melting behaviour depending on theformation of DNA heteroduplexes in the presence of a mutation. Sohomozygous mutations may be missed if only homoduplexes with the samemelting point as the wildtype homoduplexes are generated. Therefore celllines, plasmids or oligonucleotides were spiked with wildtype DNA toallow formation of heteroduplexes. According to the present disclosure,it is not necessary to spike wildtype DNA in FFPE samples because of thepresence of non-mutated stromal cells. Several homozygous mutationscomprising KRAS exon 2 G12S, G12V and G120 and the BRAF V600E mutationcan be discriminated from wildtype using HRM without spiking, whereasKRAS G12R, A59E, Q61H, Q61L and E63K as well as the PIK3CA mutationE542K can only be found in the heterozygous form.

As disclosed herein, seven different HRM assays covering the hot spotregions in four different genes (KRAS, BRAF, PIK3CA and AKT1) weredeveloped as highly specific and sensitive diagnostic tools. Thevalidation with FFPE tissues from different tumor entities showedaccurate mutation detection compared to Sanger Sequencing with a highersensitivity for HRM analysis. HRM, as a low-cost and fast method forprescreening of genomic variations represents an alternative toestablished mutation detection techniques and is therefore, as shownherein, applicable for research as well as for clinical diagnosticapproaches.

For the study underlying the present disclosure, seven different HRMassays (Table 1) have been developed as a prescreening diagnostic tool:

TABLE 1 List of the developed HRM Assays Gene exon codons KRAS 2 12, 13KRAS 3 59, 61, 63 BRAF 15 594, 600, 601 PIK3CA 7 420 PIK3CA 9 539, 542,545, 546 PIK3CA 20 1043, 1047, 1049 AKT1 2  17

As disclosed in the Example, genomic DNA isolated from cell lines,plasmids or oligonucleotides with known mutational status were used.Several rare mutations concerning KRAS exon 3 were amplified from humanFFPE tissues and cloned into the pCR-4 vector to ensure enough amount ofmutated DNA for the implementation. For each HRM assay at least threedifferent primer sets were compared and the optimal design was chosenfor the validation with genomic DNA isolated from FFPE tissues. Theprimers developed in the context of the present disclosure are disclosedin the following table 2:

TABLE 2 List of primer sets used for HRM and Sanger Sequencing PrimerSequence (5′ → 3′) AKT1-2-F CATCCCAGGCACATCTGTCC (SEQ ID NO: 1) AKT1-2-RCGCCACAGAGAAGTTGTTGAGG (SEQ ID NO: 2) AKT1-HRM-ex2-FP1GGCGAGGGTCTGACGGGTAG (SEQ ID NO: 3) AKT1-HRM-ex2-RP1GCCGCTCCTTGTAGCCAATGAAG (SEQ ID NO: 4) BRAF-15-F CTCTTCATAATGCTTGCTC(SEQ ID NO: 5) BRAF-15-R GTGAATACTGGGAACTATG (SEQ ID NO: 6) BRAF-HRM-FP3ATGCTTGCTCTGATAGGAAAATGA (SEQ ID NO: 7) BRAF-HRM-RP3ATCCAGACAACTGTTCAAACT (SEQ ID NO: 8) KRAS-12,13-FGGTGAGTTTGTATTAAAAGGTACTGG (SEQ ID NO: 9) KRAS-12,13-RGGTCCTGCACCAGTAATATGC (SEQ ID NO: 10) KRAS_HRM_FP3CCTGCTGAAAATGACTGAATATAAACTT (SEQ ID NO: 11) KRAS_HRM_RP3GCATATTAAAACAAGATTTACCTCTATTGT (SEQ ID NO: 12) KRAS-61-FCACTGTAATAATCCAGACTGTG (SEQ ID NO: 13) KRAS-61-R AATTACTCCTTAATGTCAGCTT(SEQ ID NO:14) KRAS-HRM61-FP10 ACCTGTCTCTTGGATATTCTCGA (SEQ ID NO: 15)KRAS-HRM61-RP10 ATTACTCCTTAATGTCAGCTTATTATATTCA (SEQ ID NO: 16)PIK3CA-7-F AGATATTCCCATTATTATAGAGATGATTGT (SEQ ID NO: 17) PIK3CA-7-RAGCAAATCTTCTAATCCATGAGGTA (SEQ ID NO: 18) PIK3CA-HRM-ex7-FP2GGGGAAGAAAAGTGTTTTGAAATGTG (SEQ ID NO: 19) PIK3CA-HRM-ex7-RP2ATACTAGAGTGTCTGTGTAATCAAACAAG (SEQ ID NO: 20) PIK3CA-9-FTGAAAATGTATTTGCTTTTTCTGT (SEQ ID NO: 21) PIK3CA-9-RTGTAAATTCTGCTTTATTTATTCC (SEQ ID NO: 22) PIK3CA-HRM-ex9-FP1TGACAAAGAACAGCTCAAAGCAA (SEQ ID NO: 23) PIK3CA-HRM-ex9-RP1TTTTAGCACTTACCTGTGACTCCA (SEQ ID NO: 24) PIK3CA-20.1-FTTTGCTCCAAACTGACCAA (SEQ ID NO: 25) PIK3CA-20.1-R GCATGCTGTTTAATTGTGTGG(SEQ ID NO: 26) PIK3CA-HRM-ex20-FP2 GCAAGAGGCTTTGGAGTATTTCA(SEQ ID NO: 27) PIK3CA-HRM-ex20-RP2 ATGCTGTTTAATTGTGTGGAAGATC(SEQ ID NO: 28)

Primers were, in general, designed to get an amplicon with a singlemelting peak enhancing the sensitivity of the assay. Despite testing of16 different primer sets the PCR products from the KRAS exon 3 HRM assayalways displayed two different specific melting peaks, which both werenot due to any unspecific primer dimer amplification product formation.

Since the HRM assay should be able to distinguish wildtype and mutatedDNA, all samples were additionally analysed by direct Sanger Sequencingto determine the exact mutational status. Mutational status of the celllines coincided with the literature, except KRAS exon 2 mutationanalysis for LoVo (homozygous G12V) and SW 480 (heterozygous G13D),which are just inverted in our determination (Seth, R., et al., Gut 58(2009) 1234-1241). This may be due to a mix-up of both cell lines. Thedifference in the melting curve behaviour of wildtype and mutated DNAwas visualized by normalized, temperature-shifted curves displayed asdifference plots. In each assay at least one wildtype and one mutatedcontrol DNA either isolated from cell lines, plasmids oroligonucleotides with the desired mutation were analysed.

Exemplary embodiments of the present disclosure are shown in FIG. 1. Itdisplays representative difference plots for KRAS exon 2 (FIGS. 1 A andB) and PIK3CA exon 9 (FIGS. 1 D and E) from DNA isolated from cell lines(FIGS. 1 A and D) as well as from DNA isolated from FFPE tissues (FIGS.1 B and E). The difference in the melting behaviour of heterozygousmutations results from heteroduplex formation. Heteroduplexes meltearlier and are therefore displayed above the wildtype baseline in theseillustrations (FIGS. 1 A and B). In case of homozygous mutations bothDNA strands fit to each other and depending on the nucleotide differencethere is either a different binding compared to wildtype sequence (FIG.1 A) or a binding equal to wildtype.

Concerning the KRAS exon 2 assay 4/5 homozygous mutations (G12C, G12S,G12V, G13C See FIG. 1) could be differentiated from wildtype. KRAS G12Rhomozygous mutation was only detectable after spiking with wildtype DNA.All mutations analysed for KRAS exon 3 could only be detected in theheterozygous state. The most common V600E BRAF mutation could bedifferentiated from wildtype in heterozygous as well as in homozygousform. In contrast only the heterozygous E542K PIK3CA mutation could bedifferentiated from wildtype. Concerning the E17K AKT1 mutation therewas a stronger difference between wildtype and mutation in the presenceof the heterozygous state.

Regarding that some homozygous mutations were correctly reanalysed afterspiking with wildtype DNA the concordance of HRM results and SangerSequencing for the different assays was 100%.

To determine the sensitivity of the different HRM assays dilution seriesof 100%, 50%, 25%, 12.5%, 6.3%, 3.1% and 1.6% of mutated template DNA inwildtype background were analysed. The sensitivities of the HRM assayswere 3.1% (PIK3CA exon 9, FIG. 1 F), 6.3% (KRAS exon 2, BRAF exon 15,FIG. 1 C and data not shown) and 12.5% (KRAS exon 3, PIK3CA exon 7,PIK3CA exon 20, AKT1 exon 2) of mutated DNA which could be detected in abackground of wildtype DNA.

Exclusion of BRAF, KRAS and PIK3CA Pseudogene Amplification:

Known pseudogenes are described for KRAS (McGrath, J. P., et al., Nature304 (1983) 501-506) and BRAF (Sithanandam, G., et al., Oncogene 7 (1992)795-799), spanning the complete exons with high homology to the gene,respectively and for PIK3CA, comprising exons 9-13 (Muller, C. I., etal., Leuk. Res. 31 (2007) 27-32). In case of BRAF, pseudogeneamplification could be excluded by location of primers in the intronspanning region.

In contrast, the KRAS HRM primer set for exon 12 and 13 was partiallylocated in the exon. Therefore potential pseudogene amplification due tothe high homology between KRAS pseudogene and gene needed to becomeexcluded. The KRAS HRM primer set flanks a region containing fournucleotides differing between the gene and pseudogene sequence. UsingSanger Sequencing there was no evidence of combined gene and pseudogeneamplification. A melting curve assay using hybridization probescomplementary to the KRAS gene sequence confirmed this result.

Similarly, in order to ensure a high specificity of the PIK3CA exon 9HRM assay, primer pairs were located within a region showing sequencedifferences (one nucleotide exchanged and one nucleotide deleted)between gene and pseudogene. Within the chosen amplicon there was anadditional single nucleotide exchange discriminating gene and pseudogeneamplification. As a positive control for pseudogene amplification asecond reverse primer was designed with high homology to gene andpseudogene leading to a 130 bp fragment and amplification of both.Sanger Sequencing analysis showed that PIK3CA pseudogene amplificationcould be excluded using the first gene specific primer pair whereas thesecond primer pair resulted in gene and pseudogene amplification.

Validation of the Different HRM Assays:

For the validation of the different HRM assays genomic DNA isolated fromFFPE tissues from different tumor entities (colorectal cancers,endometrial cancers, melanomas, gastrointestinal stromal tumors) wasused

In case of successful amplification by the primer pairs as disclosedabove, the analyses of KRAS exon 3, BRAF exon 15, PIK3CA exon 7, PIK3CAexon 9 and AKT1 exon 2 showed no differences in the results from eitherHRM or Sanger Sequencing. For three samples the exact mutational statusof PIK3CA exon 9 could only be determined using the HRM PCR products forsequencing analyses. Using conventional methods and primer settings thesensitivity was too low to detect these mutations. The concordance fromKRAS exon 2 HRM assay (205/208) and PIK3CA exon 20 HRM assay (192/193)with Sanger Sequencing was 99%, respectively. All analysed samples withdiscrepancy in both methods were mutated according to the HRM analyses,but wildtype using Sanger Sequencing.

HRM only allows the discrimination between wildtype and mutant samples,therefore additional Sanger Sequencing may be done when the exactmutational status is needed. KRAS exon 2 (A), 19 different KRASmutations could be either detected in codon 12 (G12D, G12V, G120, G12S,G12A, G12R), codon 13 (G13D, G130, G13_V14insG), codon 14 (V14A), codon59 (A59E), codon 60 (G600), codon 61 (Q61H: c.183A>C, Q61H: c.183A>T,Q61K; G60G, Q61L: c.182A>T, Q61L: c.182_(—)183AA>TG), codon 63 (E63K) orcodon 66 (A66A). After validation of BRAF HRM assays four differentmutations in codon 594 (D594G), codon 600 (V600E, V600K) and codon 601(K601E) were found. The validation of the PIK3CA HRM assays showed 9different mutations: codon 542 (E542K), codon 545 (E545K, E545G), codon546 (Q546K, Q546R, Q546P), codon 1043 (M10431) and codon 1047 (H1047R,H1047L). All these mutations lead to different melting behaviourcompared to wildtype DNA. No mutations were found in PIK3CA exon 7 andAKT1 exon 2.

The following table summarizes the newly identified nucleic acidsequence variations with the resulting changes in the amino acidsequence of KRAS exons 2 and 3. The nucleotide that is being changedwith respect to the wildtype sequence is underlined.

TABLE 3 Newly identified nucleic acid sequence var-iations. The underlined nucleotides representdifferences from the wild type sequence. Amino acid Seq. ID. levelDNA level NO: exon 2, codon c.40_41insGCG SEQ ID 13GTT GGA GCT GGT GGC GGC GTA NO: 31 (G13_V14insG) GGC AAG exon 2, codonc.41T > C SEQ ID 14 GTT GGA GCT GGT GGC GCA GGC NO: 32 (V14A) AAGexon3, codon60 c.179G > A SEQ ID (G60D) ACA GCA GAT CAA GAG GAG TACNO: 33 AGT exon 3, codon c.182_183AA > TG SEQ ID 61ACA GCA GGT CTG GAG GAG TAC NO: 34 (Q61L) AGT

Accordingly, in one aspect, the present disclosure is also directed tospecific hybridization probes which are capable of detecting the newlyidentified mutations. Such hybridization probes include oligonucleotidescomprising a sequence according to SEQ. ID. NO: 31, 32, 33, 34 or theirrespective complements. Typically, such hybridization probes arecompletely identical with or complimentary to the wild type sequence ofthe target DNA at all position which do not correspond to the site ofthe mutation that shall become detected.

The length of such oligonucleotides may vary from 15 to 30 nucleotideresidues, for example. These oligonucleotides are ideally completelyidentical or completely complementary to the corresponding gene,including its sequence variation which shall become detected. In someillustrative embodiments, such a hybridization probe may be labeled,e.g., with a fluorescent label. In some such embodiments, such ahybridization probe may be one member of a pair of FRET hybridizationprobes and may be used for real time PCR, as disclosed in U.S. Pat. No.6,174,670.

As described and disclosed herein, embodiments of the instant disclosureinclude a new analytic method for determining the increased likelihoodof a response to a targeted treatment of a cancer disease, comprisingthe steps of: a) isolating genomic DNA from a patient sample; b)amplifying at least one fragment of said DNA by means of PCR with aspecific pair of amplification primers; c) determining, whether saidamplified fragment has a wildtype sequence or comprises a mutation bymeans of a High Resolution Melting Analysis (HRM); and d) correlatingthe presence or absence of a mutation with an increased likelihood ofsuccess of said therapeutic treatment.

As disclosed herein, the term “targeted treatment” is defined as amedical treatment, characterized in that said medical treatment isselected from a number of different treatments available for the samephenotypic disease, such selection being based on the results ofmonitoring some previously determined biological parameter o saidpatient.

In some embodiments, subsequent to step c), the exact mutation isidentified by means of a hybridization analysis or by means ofsequencing. Hybridization analysis may be performed using thehybridization probes as disclosed above, for example. Sequencing may bedone by any of the methods available in the art. In some cases, it maybe useful for predicting therapeutic responses not only based on thefact that a certain region is mutated but it may also be important toknow what kind of mutation occurs.

Thus, in some embodiments, the inventive method disclosed herein, underthese circumstances, is a two-step process: first HRM is performed inorder to identify, whether a certain gene is mutated; and second,sequencing analysis is performed but only in case HRM has revealed thatthe gene is mutated. Since HRM is an easy, straight forward andcomparatively cheap analytical method, the number of required sequencingreactions can become reduced dramatically and the costs for drugsusceptibility testing can be significantly reduced.

According to some illustrative embodiments, the patient sample isFormalin Fixed Paraffin Embedded (FFPE) tissue. If this is the case,then High Resolution Melting Analysis may be performed without anyspiking of DNA. Since FFPE derived samples from cancer patients usuallycomprise tumor cells and non tumor cells, a hetoroduplex formationbetween wild type DNA originating from healthy tissue and mutated DNAfrom the tumor can be expected and High resolution Melting analysis canbe performed.

In illustrative embodiments of the instant disclosure, the DNA fragmentsbeing analyzed may be any selected from a group comprising KRAS, exon 2,KRAS, exon 3, BRAF exon 15, PIK3CA exon 7, PIK3CA exon 9, PIK3CA exon20, and AKT1 exon 2, for example.

For example, presence of mutations within exon 2 or exon 3 of KRAS maybe indicative for a resistance against an anti-EGFR antibody basedtherapy, especially with respect to colorectal carcinoma. Also inparticular, the presence of mutations in BRAF may be indicative of aresponsiveness to RAF inhibitors such as PLX4032, especially inmalignant melanoma for example. Furthermore, mutations within PIK, exon7, 9 or 20 or AKT1 exon 2 may be helpful for the appropriatetherapeutics of breast and lung carcinoma, for example.

The following examples, sequence listing, and figures are provided forthe purpose of demonstrating various embodiments of the instantdisclosure and aiding in an understanding of the present disclosure, thetrue scope of which is set forth in the appended claims. These examplesare not intended to, and should not be understood as, limiting the scopeor spirit of the instant disclosure in any way. It should also beunderstood that modifications can be made in the procedures set forthwithout departing from the spirit of the disclosure.

ILLUSTRATIVE EMBODIMENTS

The following comprises a list of illustrative embodiments according tothe instant disclosure which represent various embodiments of theinstant disclosure. These illustrative embodiments are not intended tobe exhaustive or limit the disclosure to the precise forms disclosed,but rather, these illustrative embodiments are provided to aide infurther describing the instant disclosure so that others skilled in theart may utilize their teachings.

1. A pair of amplification primers with oligonucleotide sequencesselected from the following group of combination of sequences:

-   -   Seq. ID. No: 1 and Seq. ID. NO: 2;    -   Seq. ID. No: 3 and Seq. ID. NO: 4;    -   Seq. ID. No: 5 and Seq. ID. NO: 6;    -   Seq. ID. No: 7 and Seq. ID. NO: 8;    -   Seq. ID. No: 9 and Seq. ID. NO: 10;    -   Seq. ID. No: 11 and Seq. ID. NO: 12;    -   Seq. ID. No: 13 and Seq. ID. NO: 14;    -   Seq. ID. No: 15 and Seq. ID. NO: 16;    -   Seq. ID. No: 17 and Seq. ID. NO: 18;    -   Seq. ID. No: 19 and Seq. ID. NO: 20;    -   Seq. ID. No: 21 and Seq. ID. NO: 22;    -   Seq. ID. No: 23 and Seq. ID. NO: 24;    -   Seq. ID. No: 25 and Seq. ID. NO: 26; and    -   Seq. ID. No: 27 and Seq. ID. NO: 28.        2. A composition comprising at least one pair of amplification        primers according to embodiment 1.        3. A kit comprising at least one pair of amplification primers        according to embodiment 1.        4. An oligonucleotide comprising a sequence selected from a        group consisting of Seq. ID. NO: 31 or its complement, Seq. ID.        NO: 32 or its complement, Seq. ID. NO: 33 or its complement, and        Seq. ID. NO: 34 or its complement.        5. A method for determining the increased likelihood of a        response to a targeted treatment of a cancer disease, comprising        the steps of:    -   i. isolating genomic DNA from a patient sample;    -   ii. amplifying at least one fragment of said DNA by means of PCR        with a specific pair of amplification primers;    -   iii. determining whether said amplified fragment has a wildtype        sequence or comprises a mutation by means of a High Resolution        Melting Analysis (HRM); and    -   iv. correlating the presence or absence of a mutation with an        increased likelihood of success of said targeted treatment,        characterized in that said at least one fragment is selected        from a group comprising of KRAS, exon 2, KRAS, Exon 3, BRAF exon        15, PIK3CA exon 7, PIK3CA exon 9, PIK3CA exon 20, and AKT1 exon        2.        6. A method according to embodiment 5, characterized in that at        least one pair of amplification primers according to claim 1 is        used.        7. A method according to any of embodiments 5-6, wherein prior        to step d) the mutation is identified by means of a        hybridization analysis or by means of sequencing.        8. A method according to any of embodiments 5-7, wherein the        patient sample is Formalin Fixed Paraffin Embedded (FFPE)        tissue.        9. A method according to embodiment 8, wherein High Resolution        Melting Analysis is performed without any spiking of DNA.

EXAMPLES Example 1 Design of HRM Assays

Cell Lines, Plasmids, Oligonucleotides and Patient Samples:

Seven different HRM assay were designed as can be seen from table 1disclosed above. For the design, the cell lines as compiled in table 4were used:

TABLE 4 Human cell lines used for the development of seven different HRMassays for KRAS exon 2; KRAS exon 3; BRAF exon 15; PIK3CA exon 7; PIK3CAexon 9; PIK3CA exon 20 and AKT1 exon 2. Used abbreviations: ex = exon,he = heterozygous, ho = homozygous, wt = wildtype PIKCA exon 7; KRASexon 2; PIK3CA exon 9; cell lines KRAS exon 3 PIK3CA exon 20; BRAF exon15 AKT1 exon 2 A549 ex2: G12S (ho) ex7: wt ex2: wt BT-20 ex7: wt ex2: wtex9: P539R (he) ex20: H1047R (he) CaCo-2 ex2: wt ex7: wt ex2: wt CaSkiex7: wt ex2: wt ex9: E545K (he) ex20: wt HCT116 ex2: G13D (he) ex7: wtex15: wt ex2: wt ex9: wt ex20: H1047R (he) HT-29 ex2: wt ex7: wt ex15:V600E (he) ex2: wt ex3: wt LoVo ex2: G12V (ho) ex7: wt ex2: wt LS174Tex2: G12D (he) ex7: wt ex2: wt ex9: wt ex 20: H1047R (he) MCF-7 ex7: wtex2: wt ex9: E545K (he) ex20: wt Mel501 ex7: wt ex15: V600E (he) ex2: wtMia PaCa-2 ex2: G12C (ho) ex7: wt ex2: wt NCI H460 ex3: Q61H ex7: wtex2: wt (ho, c.183A > T) ex9: E545K (he) ex20: wt NCI N417 ex7: wt, ex2:wt ex9: Q546K (he) ex20: wt RPMI 8226 ex2: G12A (he) ex7: wt ex2: wtOAW-42 ex7: wt ex2: wt ex9: wt ex20: H1047L (he) Sk-Mel28 ex7: wt ex15:V600E (ho) ex2: wt SW 480 ex2: G13D (he) ex7: wt ex2: wt SW 620 ex2:G12V (ho) ex7: wt ex2: wt SW 948 ex7: wt ex2: wt ex9: E542K (ho) ex20:wt T47D ex7: wt ex2: wt ex9: wt ex20: H1047R (he)

All cell lines were grown under standard conditions. Determination ofmutation status was done by Sanger Sequencing. Cell lines withoutdescription of mutation status were not determined.

Plasmids with human genomic DNA fragments containing KRAS mutations wereeither obtained from Roche (Pleasanton, Calif., USA) or generated bycloning the relevant fragments into the pCR-4 vector by using the TOPOTA Cloning Kit for Sequencing (Invitrogen, Karlsruhe, Germany) accordingto manufacturer's instructions. For rare mutations (AKT1: E17K, PIK3CA:C420R), whole PCR products were ordered (Metabion, Martinsried,Germany).

The HRM mutation assays were validated on routinely used FFPE tissues(colorectal cancers, endometrial cancers, melanomas, gastrointestinalstromal tumors). Analysed sample numbers ranged from 131 to 205depending on the assay. Ethical approval for this research was obtainedfrom the local ethical committee.

Genomic DNA from cell lines was isolated using the QIAamp DNA Mini Kit(Qiagen, Hilden, Germany) according to manufacturer's protocols. For DNAisolation from FFPE tissue, a tumor area was marked on the hematoxylinand eosin (H&E) stained sections and macrodissected on correspondingunstained 10 μm thick slides for subsequent DNA isolation. DNApurification was performed using the BioRobot M48 Robotic Workstationand the corresponding MagAttract DNA Mini M48 Kit (Qiagen) following themanufacturer's instructions. DNA quantity was assessedspectrophotometrically using the NanoDrop ND 1000 (Peqlab, Erlangen,Germany) and quality of genomic DNA was confirmed by agarose gelelectrophoresis. Extracted DNA was temporary stored at 4° C. andlong-term stored at −20° C. or −80° C. High Resolution Melting Analyses(HRM). HRM analyses were performed on a LightCycler™ 480 (RocheDiagnostics, Mannheim, Germany). Each run included mutated and wildtypeDNA as controls as shown in tables 4 and 5.

TABLE 5 Plasmids used for the development of KRAS HRM assays and meltingcurve analysis by using hybridization probes to exclude any KRASpseudogene amplification. Plasmids KRAS mutation status Reference pCR-4E63K 27 E63K (c.187G > A) this work pCR-4 A59E 35 A59E (c.176C > A) thiswork pCR-4 Q61H (CAC) A27 Q61H (c.183A > C) this work pCR-4 Q61L (CTA)B6 Q61L (c.182A > T) this work pCR-4 Q61L (CTG) C3 Q61L (c.182_183AA >this work TG) pK12C1 G12R (c.34G > C) Roche (Pleasanton, CA, USA) pK13T1G13C (c.37G > T) Roche (Pleasanton, CA, USA)

For each assay at least three different primer sets (HPLC purified,Sigma Aldrich, Munich, Germany) were selected to flank the hot spotmutation regions of the gene. Amplicon lengths ranged from 100 bp to 186bp depending on the HRM assay. All designed primers are compiled intable 2 as disclosed in the specification above.

Each reaction mixture contained 10 ng of genomic DNA or 10 fg of plasmidDNA or 0.2-2 fM oligonucleotides, 200 nmol/L of each primer, 10 μl ofLightCycler LC480 High Resolution Melting Master (Roche Diagnostics),3.5 mmol/L MgCl₂ or 3.0 mmol/L MgCl₂ in the case of PIK3CA exon 20 andAKT1 exon 2 assay and A. dest. to a final volume of 20 μl. All reactionswere routinely performed in triplicates. PCR and melting curveconditions were used according to manufacturer's instructions. Annealingtemperatures were 60° C. for all HRM assays except the following: BRAFexon 15 (59° C.), KRAS exon 3 (58° C.) and AKT1 exon 2 (63° C.). Themelting curves were analysed by Gene Scanning software (RocheDiagnostics) with normalized, temperature-shifted curves displayedfinally as a difference plot. Either genomic wildtype DNA isolated fromcell lines or plasmid DNA was used for normalization.

In case of significant differences of the fluorescence level for alltriplicates, which were not in the range of variation detected forwildtype control, samples were considered as mutated. The HRM methoddepends on heteroduplex formation, so statistically rare homozygousmutations might not be detected. For this purpose, all control DNAsamples negative on initial screening were spiked with wildtype DNA andreanalysed. FFPE tissues were not spiked with wildtype DNA as aftermacrodissection usually few normal tissue and/or stromal cells areincluded and allow heteroduplex formation. To determine the sensitivityof HRM assays mutated DNA or oligonucleotides containing the mutation ofinterest were serially diluted with wildtype DNA.

Sanger Sequencing.

Sanger Sequencing analyses were performed from all samples to determinethe mutation status and to verify the HRM results. Depending on the HRMassay design, verification of results were either done fromconventionally amplified DNA fragments using Platinum Taq DNA polymerase(Invitrogen) or from fragments amplified by real-time FOR for HRManalysis. The latter PCR products could be directly used for sequencinganalyses, whereas conventionally amplified PCR products were checked forthe right fragment length by agarose gel electrophoresis and purifiedusing S-300 HR MicroSpin Columns (Amersham Pharmacia Biotech, Freiburg,Germany) or polyethylene glycol precipitation. Purified PCR productswere sequenced using BigDye Terminator v1.1 Cycle Sequencing kit.Sequences were run on an ABI Prism 3130 automated sequencer and datawere manually edited using Sequencer analysis software (all AppliedBiosystems, Darmstadt, Germany).

Melting Curve Analysis by Using Hybridization Probes.

A melting curve assay using hybridization probes was designed to excludeKRAS pseudogene amplification. The Hybridization probes used were:

-   -   KRAS1, which was a 3′ Fluorescein probe, comprising the        nucleotide sequence:

(SEQ ID NO: 29) TAGGCAAGAGTGCCTTGACGA, and

-   -   KRAS2 which was 5′ Red640 labeled probe and a 3′terminal        phosphate block, comprising the nucleotide sequence:

(SEQ ID NO: 30) ACAGCTAATTCAGAATCATTTTGTGGAC GAATATGATCCA.

The probes were designed with the LightCycler Probe Design 2.0 software(Roche Diagnostics) and tested for their specificity as described above.An asymmetric PCR was performed by using LightCycler Probes Master(Roche Diagnostics) according to manufacturer's instructions. Briefly,10 ng genomic template DNA, 10 fg plasmid DNA or 0.2 μM KRAS pseudogenecontrol were mixed with 0.2 μM hybridization probes, 0.075 μMKRAS_HRM_FP3, 0.5 μM KRAS_HRM_RP3 and A. dest. to a final volume of 20μl. The whole PCR products for the KRAS pseudogene control were acquiredby purchase (Metabion). PCR and melting curve conditions were usedaccording to manufacturer's instructions.

All references cited in this specification are herewith incorporated byreference with respect to their entire disclosure content and thedisclosure content specifically mentioned in this specification.

While this disclosure has been described as having an exemplary design,the present disclosure may be further modified within the spirit andscope of this disclosure. This application is therefore intended tocover any variations, uses, or adaptations of the disclosure using itsgeneral principles. Further, this application is intended to cover suchdepartures from the present disclosure as come within the known orcustomary practice in the art to which this disclosure pertains.

1. (canceled)
 2. A method of identification of hot-spot mutations in theKRAS gene in a sample, the method comprising the steps of: i. isolatinggenomic DNA from the sample; ii. amplifying a fragment of said DNA bymeans of PCR with a specific pair of amplification primers; iii.determining, that said amplified fragment comprises a mutation in theKRAS gene by means of a High Resolution Melting Analysis (HRM); and iv.performing DNA sequencing to determine that the mutation is one of themutations G13_V14insG, V14A, or G60D in the KRAS gene.
 3. The method ofclaim 2, wherein in step ii, the amplification primers comprise one ormore sequences selected from the group consisting of SEQ ID NOs: 1-28.4. (canceled)
 5. The method of claim 2, wherein the sample is FormalinFixed Paraffin Embedded (FFPE) tissue.
 6. The method of claim 5, whereinsaid step of determining comprises performing High Resolution MeltingAnalysis without any spiking of DNA.
 7. The method of claim 2, whereinsaid step of determining comprises comparing HRM analysis results of theamplified fragment to a control.
 8. The method of claim 7, wherein thecontrol comprises HRM analysis results of a wildtype sample. 9.(canceled)
 10. The method of claim 2, wherein HRM is performed using aprobe selected from a group consisting of Seq. ID. NO: 31 or itscomplement, Seq. ID. NO: 32 or its complement, Seq. ID. NO: 33 or itscomplement, and Seq. ID. NO: 34 or its complement.